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DISEASE IN WILDLIFE OR EXOTIC SPECIES

Dermatophytosis caused byTrichophytonspp.in a Tenerife Lizard (Gallotia galloti): an

Immunohistochemical Study

J. Oros*, J. D. Hernandez

, J. Gallardo*, P. Lupiola* and H. E. Jensen

* Veterinary Faculty, University of Las Palmas de Gran Canaria (ULPGC), Trasmontana s/n, 35416 Arucas, Serviexotic,

Vega de San Mateo, Las Palmas, Spain andDepartment of Pharmacology and Pathobiology, The Royal Veterinary and

Agricultural University, 13 Bulowsvej, DK-1870 Frederiksberg C, Copenhagen, Denmark

Summary

Reports of dermatophytosis in reptiles are rare. This report describes the microscopical and immunohistochem-ical findings in a case of dermatophytosis caused by Trichophytonspp. in a 2-year-old Tenerife lizard (Gallotiagalloti) with ulcerative and pustular skin lesions. Microscopically, the lesions were characterized by superficialepidermal pustules containing heterophils with numerous fungal hyphae that stained by periodic acideSchiffand Grocotts stain.Fungal culture was not performed, but a panel of polyclonal antibodies specific for differentfungal genera was applied to tissue sections. These immunohistochemical studies demonstrated reactivity of thehyphae only with antiserum specific for Trichophytonspp.

2012 Elsevier Ltd. All rights reserved.

Keywords: dermatophytosis; immunohistochemistry; Tenerife lizard; Trichophyton spp.

The term dermatophytosis describes infection by kera-tinophilic fungi of the generaMicrosporum,Trichophytonand Epidermophyton (Pare and Jacobson, 2007). Derma-tophytes are rarely isolated from reptiles (Hazellet al.,1985; Miller et al., 2004) and it has been suggestedthat there is insufficient evidence to support the occur-rence of dermatophytosis in these animals (Pareand

Jacobson, 2007).Fungal infections in reptiles are often diagnosed by

culture or histopathology, but in the absence of culture,

immunohistochemistry (IHC) provides an alternativediagnostic approach. Reports of immunohistochemicaldiagnosis of mycotic infections in reptiles are scarce(Oros et al., 2004a,b; 2011). The present report de-scribes a case of dermatophytosiscaused by Trichophytonspp. in a Tenerife lizard (Gallotia galloti).

A captive 2-year-old, 205 g, female Tenerife lizard,developed several irregular pustular and ulcerativecutaneous lesions (0.1 cm diameter) in the interscap-ular region. The lizard had been taken from the wild

from Vi~na de las Arenas (Tenerife) when it was youngbecause it had lost its right hindlimb after a cat attack.The lizard had been kept alone in a 60 50 50 cmterrarium with artificial grass as substrate and corkbark sheets as hiding places. The diet consisted ofcrickets coated with a vitamin/mineral powder andsome vegetables and grapes. Using a commercialheat mat and sunlight for a basking site the ambienttemperature in the terrarium was maintained at24e28C. Due to financial constraints, haematologi-

cal examination and microbiological cultures werenot performed. The lizard was treated with enroflox-acin (Baytril, Bayer, Leverkusen, Germany) at15 mg/kg orally q24h for 21 days and 1% chlorhexi-dine solution (Menalmina, Orravan S. L., Sant

Joan Desp, Spain) was applied topically. The healthof the lizard declined, with the animal developinglethargy and inappetence before dying.

At necropsy examination, the lizard was cachectic,with muscle atrophy. Several irregular pustular andulcerative skin lesions (0.3 cm diameter) were ob-served in the interscapular region (Fig. 1). Bothkidneys contained numerous round white fociCorrespondence to: J. Oros (e-mail: joros@dmor.ulpgc.es).

0021-9975/$ - see front matter 2012 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.jcpa.2012.11.245

J. Comp. Path. 2013, Vol. 149, 372e375 Available online atwww.sciencedirect.com

www.elsevier.com/locate/jcpa

mailto:joros@dmor.ulpgc.eshttp://dx.doi.org/10.1016/j.jcpa.2012.11.245http://dx.doi.org/10.1016/j.jcpa.2012.11.245http://dx.doi.org/10.1016/j.jcpa.2012.11.245http://www.sciencedirect.com/science/journal/00219975http://www.elsevier.com/locate/jcpahttp://www.elsevier.com/locate/jcpahttp://www.sciencedirect.com/science/journal/00219975http://dx.doi.org/10.1016/j.jcpa.2012.11.245http://dx.doi.org/10.1016/j.jcpa.2012.11.245http://dx.doi.org/10.1016/j.jcpa.2012.11.245mailto:joros@dmor.ulpgc.es

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(1e2 mm diameter). No gross lesions were present inother organs. Tissue samples from all major organswere fixed in 10% neutral buffered formalin, pro-cessed routinely and embedded in paraffin wax.Sections (4 mm) were stained with haematoxylinand eosin (HE). Selected samples were also stainedwith periodic acideSchiff (PAS) and Grocottsmethenamine silver nitrate (GMS) stain.

Microscopically, there was severe superficial epi-dermal pustule formation and these pustules con-tained mainly heterophils. Secondary erosion andulceration was observed in some areas. NumerousPAS- and GMS-positive fungal hyphae were associ-ated with the lesions. These hyphae were branched,septate and approximately 1.4e3.6 mm in diameter.

Some hyphae were present in the dermis wherethey were associated with an inflammatory infiltra-tion of macrophages and lymphocytes. A discretenumber of small granulomata were also present inthe dermis and these contained some hyphae inthe central necrotic areas. No bacterial colonieswere observed. Both kidneys showed numerous ur-ate tophi associated with a mixed inflammatory re-action. No histological lesions were detected inother major organs.

Because fungal cultures were not taken at the timeof necropsy examination, an immunohistochemicalstudy was carried out in order to determine the iden-tity of the organism. Sections of the cutaneous lesionswere mounted on adhesive slides (Superfrost Plus,

Menzel-Glaser, Braunschweig, Germany) and keptat 4C until processed. Primary reagents for immuno-labelling included monoclonal antibodies specific forantigens of Aspergillus spp. (WF-AF-1; Dako,Glostrup, Denmark) and the Mucorales group(WSSA-RA-1; Dako) and rabbit polyclonal anti-bodies specific for Candida spp. and Trichophyton spp.(Trichophyton verrucosum and Trichophyton mentagro-

phytes) (Jensen et al., 1996). Genus-specific rabbitpolyclonal antibodies raised against Aspergillus fumiga-tus,Aspergillus flavus, Aspergillus niger,Geotrichum candi-dum, Fusarium solani and Scedosporium apiospermum

were also applied (Jensenet al., 1996). All polyclonalantibodies were absorbed against other fungal organ-isms (Okuda et al., 1987;Jensen et al., 1996). The spec-ificity and dilutions of primary antibodies aresummarized inTable 1. A commercial detection sys-tem (Power-vision+ Poly-HRP Histostaining Kit;Immunovision Technologies Co., Burlingame,California, USA) was used to visualize immunolab-elling. Application of this detection system wasfollowed by incubation for 6 min with 3-amino-9-ethyl-carbazole (AEC) solution. The sections werecounterstained with Harriss haematoxylin for10 sec. Tissues from mice and rabbits infected exper-

imentally with reference fungi were used as positivecontrols (Jensenet al., 1996). In addition, cutaneouslesions induced experimentally in two green iguanas(Iguana iguana) byChrysosporiumanamorph ofNanniz-ziopsis vriesii(University of Alberta Microfungus Col-lection and Herbarium UAMH number 7583) wereused as negative tissue controls.

The fungal elements within the cutaneous lesionswere labelled only by the antiserum specific forTrichophyton spp. (Fig. 2). No immunolabelling was

Fig. 1. Interscapular region of a Tenerife lizard (Gallotia galloti)showing pustular and ulcerative skin lesions (arrow).

Table 1

Specificity and dilution of primary antibodies used in IHC

Antibody Fungal species Fungal specificity Dilution

Monoclonal Aspergillusspp. Aspergillusspp. 1 in 2

Monoclonal Mucorales (Zygomycetes) Mucorales (Zygomycetes) 1 in 4

Polyclonal Trichophyton mentagrophytes Trichophytonspp. 1 in 600

Polyclonal Trichophyton verrucosum Trichophytonspp. 1 in 600

Polyclonal Aspergillus fumigatus Aspergillusspp. 1 in 64

Polyclonal Candida albicans Candida spp. 1 in 256

Polyclonal Geotrichum candidum Geotrichum candidum 1 in 32

Polyclonal Fusarium solani Fusariumspp. 1 in 16

Polyclonal Scedosporium apiospermum Scedosporiumspp. 1 in 16

Dermatophytosis in a Tenerife Lizard 373

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observed in the tissues from iguanas infected experi-mentally withChrysosporiumanamorph ofN. vriesii.

There are no reports ofTrichophyton spp. causingskin disease or systemic mycotic infection in reptilesof the family Lacertidae and reports of dermatophyto-sis in reptiles are rare. Trichophyton terrestre was isolatedfrom lesions in a group of blue-tongued skinks (Tiliquascincoides) with progressive digital necrosis (Hazellet al., 1985), but causality was unclear (Pare and

Jacobson, 2007). In addition, the recognition overthe last decade of the Chrysosporium anamorph of

N. vriesii as a major cause of hyalohyphomycosis inreptiles (Pare et al., 1997, 2006;Abarca et al., 2009;Hellebuycket al., 2010), casts some doubt on the au-thenticity of previously reported cases of infectionwith Trichophyton spp., Geotrichum spp., Chrysosporiumspp.,Malbranchea spp. and Trichosporonspp., as thesefungi may be readily confused with each other (Pareand Jacobson, 2007).

However,Trichophyton spp. was isolated from twogreen anacondas (Eunectes murinus) with dermallesions (Miller et al., 2004) and recently, IHC wasused to identify Trichophytonspp. associated with sys-

temic lesions in an olive ridley sea turtle (Lepidochelysolivacea) (Oroset al., 2011). The positive immunolab-elling of the fungal elements in this lizard with anti-bodies specific for Trichophyton spp. and the absenceof immunoreaction when using these antibodies ontissues containing the Chrysosporium anamorph ofN. vriesii, confirms the specificity of the antibodiesand the aetiology of the mycotic infection.

The origin of the fungal infection in this lizardremains unknown. The genus Trichophyton includesanthropophilic, zoophilic and geophilic species, butcross infections are frequent (Oros et al., 2011). Super-ficial dermatomycoses occur when the skin comes in

contact with spores or conidia of pathogenic fungithat colonize the outer epidermal layers. Invasion ofthe fungal hyphae into the stratum spinosum andstratum germinativum triggers a heterophilic re-sponse, typically, but not always, leading to coagula-

tive and liquefactive necrosis of the epidermissurrounding the fungal elements (Pare and

Jacobson, 2007).The diagnosis of cutaneous or systemic infections in

reptiles is usually achieved by culture or histopathol-ogy. More recently, molecular techniques for fungalidentification from pure cultures have been developed(Borman et al., 2008). However, a firm diagnosis ofmycosis can never rely solely on isolation of a fungusfrom clinical material and can only be established iffungal elements are demonstrated histologicallywithin the lesions and inflammation is present around

the fungal elements. IHC has proven to be a powerfultool for the accurate diagnosis of a number of impor-tant mycoses in man and domestic animals (Jensenet al., 1996) and in sea turtles (Oros et al., 2004a,b;2011), especially when cultures are not available.

This case suggests that Trichophytonspp. should beincluded as a possible aetiology in the differential di-agnosis of cutaneous mycoses in lizards. In addition,exotic animal veterinarians should be aware of theavailability of immunohistochemical techniques foridentifying fungi in reptiles.

References

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Fig.2. Specific immunolabelling ofTrichophyton spp. inthe cutane-ous lesions from a Tenerife lizard. Bar, 58 mm.

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Received, September 24th, 2012Accepted, November 29th, 2012

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